ABSTRACT There are several models, partially overlapping and partially conflicting, of how stem cells of the adult brain maintain their pool, divide, and give rise to neuronal and glial cells. Resolving those modes is important because they imply different long-term consequences for the cognitive function, effects of stress and disease, and response to therapies. Depending on the model, these consequences range from the continuous support of the stem cell pool and their ability to generate neuronal and glial progeny to the exhaustion of the stem cell pool and cessation of the ability to produce progeny. Partially, the debate about the basic scheme of the stem cell life cycle is explained by inherent limitations of the approaches employed to study this issue, which are now limited to nucleotide labeling of division events, clonal analysis, or live observation. Here we propose endogenous barcoding as an orthogonal approach and describe experiments to assess its feasibility for studying stem cells and generation of neurons and glia. This approach is based on Polylox, a new Cre recombinase-driven DNA recombination substrate introduced into the mouse germline. Cre induces random recombination of nine unique DNA elements, creating over a million distinct codes and uniquely marking cells that have supported recombination of the Polylox allele and all of their progeny. We propose to induce the recombination events in neural stem cells of the adult hippocampus of compound lines carrying the Polylox allele and determine the overall composition of their progeny. Furthermore, we propose to combine the Polylox endogenous barcoding approach with single cell transcriptomics to determine the profiles of individual barcoded cells and their position on the trajectory from stem cells to differentiated neurons or glia. Thus, in our first specific aim we will generate multiallelic transgenic mouse lines carrying a combination of the Polylox transgene with transgenes for stem cell- specific Cre recombinase and for lineage markers and will then assess and isolate hippocampal cells carrying particulars barcode and deduce their relation. In our second specific aim, we will generate additional multiallelic lines carrying Polylox barcode cassette, apply recombination-induced endogenous barcoding, and then use single-cell transcription analysis combined with barcode analysis as a novel approach for determining division, differentiation, and lineage of individual neural stem cells. Both approaches will also help to resolve some of the unanswered or contradictory questions about the models of stem cell maintenance and division and generation of glial and neuronal progeny. Our exploratory project will introduce a new modality in the studies of neural stem cells, neurons, and glia and will serve as a platform for further studies of dynamic regulation of the stem cell life cycle in the developing and adult nervous system.